System and method for organoid culture

ABSTRACT

The present disclosure provides a system, including methods and apparatus, for culturing, monitoring, and/or analyzing organoids. In an exemplary method of organoid culture, the method may comprise disposing a scaffold in a receptacle having an open side. A sealing member may be bonded to the open side of the receptacle to create a chamber. An organoid may be formed in the chamber using the scaffold. Fluid and/or at least one substance may be introduced into the chamber from an overlying reservoir for contact with the organoid.

An organoid (a “mini-organ”) is a three-dimensional mass of cells ofdifferent types produced in vitro and having some resemblance to anorgan, such as exhibiting a realistic histology of organ-specifictissue. The mass of cells can be generated by seeding a matrix with asmall number of stem cells. The stem cells then proliferate,differentiate, and self-organize within the matrix, while using thematrix as a scaffold. With this approach, organoids resembling tissuefrom the brain, heart, intestine, kidney, liver, and stomach, amongothers, have been generated so far. These promising results suggest thatorganoid culture has the potential to provide new insights into organdevelopment and function, and to recapitulate disease models that allowdrug screening in vitro. Organoids may revolutionize how drugs arediscovered and medicine is personalized.

There are a number of problems that limit the ability of researchers toexploit organoids fully. First, as organoids increase in size, they mayneed to be fed from both the inside and the outside, which presents achallenge. Second, each different type of organoid can require anoptimized three-dimensional matrix scaffold structure, media exchange(feeding) appropriate for that structure, and possibly even the abilityto experience mechanical resistance or controlled force, to allow propergrowth and development of a functioning organoid. Third, there are novessels optimized for exposing large organoids to different types ofreagents to allow screening. Fourth, there are no vessels optimized formonitoring large organoids in situ by imaging methods. Instead,organoids are often imaged after they have been fixed and sectionedphysically.

Accordingly, systems and methods are needed for improving organoidculture, such as by providing the ability to efficiently grow, monitor,and analyze large organoids.

SUMMARY

The present disclosure provides a system, including methods andapparatus, for culturing, monitoring, and/or analyzing organoids orother organized multi-cellular structures. In an exemplary method oforganoid culture, the method may comprise disposing a scaffold in areceptacle having an open side. A sealing member may be bonded to theopen side of the receptacle to create a chamber. An organoid may beformed in the chamber using the scaffold. Fluid and/or at least onesubstance may be introduced into the chamber from an overlying reservoirfor contact with the organoid.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a schematic side view of an exemplary vessel for forming,culturing, monitoring, and analyzing an organoid, taken with the vesselholding an organoid submerged in a liquid culture medium, and with frontand back walls of the vessel not visible.

FIG. 2 is an exploded side view of the vessel of FIG. 1 taken while thevessel is empty.

FIG. 3 is a side view of a body of the vessel of FIG. 2 , taken inisolation and inverted with respect to FIG. 2 before performance of amethod of culturing, monitoring, and/or analyzing an organoid in thevessel, as illustrated in FIGS. 4-10 .

FIGS. 4 and 5 are side views of the vessel body of FIG. 3 , takenrespectively during and after creation of a scaffold in a receptacle ofthe vessel by 3D printing or pipetting.

FIG. 6 is a side view of the vessel body of FIG. 5 , taken after apre-made sealing member has been bonded to the vessel body to seal anopen side of the receptacle to produce a chamber containing thescaffold.

FIG. 7 is a side view of the vessel body and sealing member of FIG. 6 ,taken after the vessel body has been flipped over to anorganoid-culturing orientation, at least one liquid medium has beenintroduced into the chamber containing the scaffold and into overlyingreservoirs that are in fluid communication with the chamber, and a lidof the vessel has covered the open tops of the reservoirs.

FIG. 8 is a side view of the vessel of FIG. 7 taken after proliferationand differentiation of stem cells in the scaffold to generate anorganoid.

FIG. 9 is a side view of the vessel of FIG. 8 taken afterremodeling/replacement of the scaffold by the developing organoid.

FIG. 10 is a side view of the vessel of FIG. 9 taken as the organoid isbeing imaged by light-sheet microscopy.

FIGS. 11-13 are side views of the vessel body and scaffold of FIG. 5illustrating an alternative approach to sealing an open side of thereceptacle of the vessel body to produce a chamber, by forming a sealingmember in situ in the receptacle.

FIG. 14 is a side view of the vessel body, sealing member, and scaffoldof FIG. 13 taken in the presence of an imaging objective, and after thevessel body has been flipped over to an organoid-culturing orientation,at least one liquid medium has been introduced into the chambercontaining the scaffold and into overlying reservoirs in fluidcommunication with the chamber, and a lid of the vessel has covered theopen tops of the reservoirs.

FIG. 15 is a side view of the vessel body and scaffold of FIG. 12illustrating, relative to FIG. 13 , a modified approach to sealing anopen side of the receptacle of the vessel body to produce a chamber, byforming a sealing member in situ in the receptacle.

FIG. 16 is a schematic side view of an exemplary vessel strip forforming, culturing, monitoring, and/or analyzing an array of organoids,where the vessel strip has a set of vessels connected directly to oneanother.

FIG. 17 is a schematic side view of the vessel strip of FIG. 16 , takenduring culture of organoids and illustrating an exemplary pumpconfiguration to transfer a liquid medium between reservoirs within eachvessel of the vessel strip.

FIGS. 17A and 17B show schematic side views of the vessel strip of FIG.16 , taken during culture of organoids and illustrating how tilting thevessel strip in opposite rotational directions can drive flow inrespective opposite directions within each chamber.

FIG. 18 is a schematic side view of another embodiment of an exemplaryvessel strip having an array of vessels, taken during culture oforganoids and illustrating an exemplary pump configuration to transfer aliquid medium among reservoirs of different vessels of the vessel strip.

FIG. 19 is a view of an exemplary embodiment of a vessel body for thevessel of FIG. 2 .

FIG. 20 is a front view of the vessel body of FIG. 19 .

FIG. 21 is a view of the vessel body of FIG. 19 , taken with the vesselbody inverted and with a pre-made sealing member exploded from areceptacle of the vessel body.

FIG. 22 is a bottom view of the vessel body of FIG. 19 , taken generallyalong line 22-22 of FIG. 20 .

FIG. 23 is a top view of the vessel body of FIG. 19 , taken generallyalong line 23-23 of FIG. 20 .

FIG. 24 is a sectional view of the vessel body of FIG. 19 , takengenerally along line 24-24 of FIG. 23 .

FIG. 25 is another sectional view of the vessel body of FIG. 19 , takengenerally along line 25-25 of FIG. 23 .

FIG. 26 is a fragmentary sectional view of another embodiment of avessel body, taken generally as in FIG. 25 , except showing only a lowerportion of the vessel body that differs from that of FIG. 25 .

FIG. 27 is a fragmentary sectional view of the vessel body of FIG. 26taken after a porous tube has been formed by 3D printing betweenchannels of the vessel body.

FIG. 28 is a sectional view of yet another embodiment of a vessel body,taken as in FIG. 25 , with the vessel body including an electromagnetoperatively associated with the receptacle of the vessel body.

FIG. 29 is a fragmentary view of the vessel body of FIG. 28 bonded to asealing member to create a chamber, with a scaffold connected to aceiling of the chamber, and with cells located on a floor of thechamber, the cells being ferromagnetic, and the electromagnet turnedoff.

FIG. 30 is another fragmentary view of the vessel body, sealing member,scaffold, and cells of FIG. 29 , taken with the electromagnet turned onsuch that the ferromagnetically-labeled cells have been pulled into thescaffold by magnetic force.

FIG. 31 is a top view of an exemplary rack to hold a linearly-arrangedset of vessels as a strip during scaffold creation and organoidformation and culture, where each vessel includes a vessel bodycorresponding to the embodiment of FIG. 19 .

FIG. 32 is a top view of the rack of FIG. 31 assembled with a set ofvessel bodies corresponding to the embodiment of FIG. 19 to create astrip.

FIG. 33 is a side view of the strip of FIG. 32 .

FIG. 34 is a top view of another exemplary rack to hold atwo-dimensional array of vessel bodies corresponding to the embodimentof FIG. 19 to create a strip.

FIG. 35 is a sectional view of still yet another embodiment of a vesselbody for the vessel of FIGS. 1 and 2 , taken generally as in FIG. 25(for a related embodiment), with an open bottom end of the vessel bodysealed with a sealing member to form a chamber, with the vessel bodydefining an access tube that is closed at its bottom end by a breachablebarrier, and with a magnet inserted into the access tube to place aworking end of the magnet adjacent (and above) the breachable barrier.

FIG. 36 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by a needle having a tip that has pierced the breachablebarrier and entered the organoid.

FIG. 37 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by a sensor/electrode having a sensing/stimulating endregion that has pierced the breachable barrier and entered the organoid.

FIGS. 38-40 are fragmentary sectional views of the vessel body of FIG.35 , taken around the breachable barrier before, during, and after a tipof a breaching instrument passes through the barrier and into thechamber from the access tube.

FIG. 41 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken generally as in FIG. 24 (for a related embodiment), suchthat three access tubes are visible, with breaching instruments disposedin the three access tubes, and with only one of breaching instrumentsextending into the chamber below the access tubes.

FIGS. 42-44 are fragmentary sectional views of a modified embodiment ofthe vessel body of FIG. 35 in which the breachable barrier is configuredto tear at a frangible web, with the views taken around the breachablebarrier before, during, and after the tip of a breaching instrumentpasses through the breachable barrier from the access tube.

FIG. 45 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by an Attenuated Total Reflectance (ATR) fiberopticprobe having a tip that has pierced the breachable barrier and enteredthe organoid.

FIG. 46 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by an imaging fiberoptic probe having a front lens thathas passed through the breachable barrier and entered the organoid.

FIG. 47 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by an illumination probe having as distal end that hasentered the organoid.

FIG. 48 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken with an organoid present in the chamber and with themagnet replaced by a pneumatic device having an expandable balloonlocated inside the organoid to create internal mechanical strain.

FIG. 49 is a sectional view of the vessel body and sealing member ofFIG. 35 , taken as in FIG. 24 (for a related embodiment) with anorganoid present in the chamber and with the magnet replaced by a pairof pneumatic devices having expandable balloons located outside theorganoid to create external mechanical strain.

FIGS. 50 and 51 are views of the vessel body and sealing member of FIG.35 , taken generally as in FIGS. 25 and 24 , respectively (for a similarembodiment), with a pair of magnets extending into the chamber viacorresponding access tubes and mounting a pre-manufactured scaffoldstructure or biochip to the magnets via magnetic attraction.

FIGS. 52 and 53 are sectional views of the vessel body and sealingmember of FIG. 35 , taken with a cap mounted to and hermetically sealingthe top of the vessel body and providing a flexible member(s) that isbeing deformed by external pressure applied alternately over a pair ofreservoirs to drive liquid through the chamber alternately in oppositedirections.

DETAILED DESCRIPTION

The present disclosure provides a system, including methods andapparatus, for culturing, monitoring, and/or analyzing organoids orother organized multi-cellular structures, such as developing orfully-developed multi-cellular organisms, tissue biopsies, and/orprimary patient material, among others. In an exemplary method oforganoid culture, the method may comprise disposing a scaffold in areceptacle having an open side. A sealing member may be bonded to theopen side of the receptacle to create a chamber. An organoid may beformed in the chamber using the scaffold. Fluid and/or at least onesubstance may be introduced into the chamber from an overlying reservoirfor contact with the organoid.

The present disclosure describes a vessel for formation, culture,monitoring, and/or analysis of organoids or other organizedmulti-cellular structures, such as developing or fully developedmulti-cellular organisms, tissue biopsies, and/or primary patientmaterial, among others. The vessel may be consumable (i.e., disposableafter a single use) and/or may have a standard shape. The vessel mayinclude a vessel body defining a receptacle having an open side. Asealing member may be attached (e.g., bonded) to the vessel body to forma chamber from the receptacle. The sealing member may be pre-formed orformed in the receptacle, among others.

A three-dimensional (3D) structure (i.e., at least one matrix, which maybe a scaffold) may be disposed in the receptacle before the open sidethereof is sealed. The 3D structure may be formed in the receptacle by a3D printer. The 3D printer may dispense one or more solidifiable bioinksthat may be mixed with cells before or as the ink(s) is deposited togenerate the 3D structure. Alternatively, the cells may be introducedinto the receptacle or chamber separately from the 3D structure. Inother embodiments, the 3D structure may be formed at least partially orcompletely outside the receptacle and then disposed therein.

Suitable cells that may be introduced into the matrix scaffold as it isbeing created or after it has been created may includenon-differentiated stem cells, stem cells that have alreadydifferentiated and will continue to differentiate, cell aggregates,small organoids, and/or the like.

The 3D printer also may be utilized to apply adhesive and/or sealingfluid to the vessel body to allow sealing of the open side of thereceptacle with a transparent sealing member at the end of the printingprocess. Exemplary printing techniques that may be suitable fordispensing matrix components include droplet-based bioprinting using abioink(s), or laser-based bioprinting (e.g., laser-based direct-writingto print cells, enzymes, etc., by laser-induced forward transfer (LIFT)with a pulsed laser).

The scaffold and/or other matrix may be provided by one or morehydrogels. Each hydrogel may include one or more thermoplasticstructural components, such as Matrigel, alginate, nanofibrillarcellulose, collagen, fibrin, and/or polyethylene glycol, among others,that cooperatively form a matrix in a temperature-dependent fashion.

In some embodiments, two or more different hydrogels/matrices may bedisposed in a receptacle. The hydrogels/matrices may differ for anysuitable parameters, such as melting temperature, resistance to enzymedegradation, solubility, cell-attraction and/or cell-repulsioncharacteristics, and/or the like.

Each hydrogel/matrix may include any suitable components. Exemplarycomponents include one or more polysaccharides (e.g., glycosaminoglycans(GAGs, such as chondroitin sulfate, dermatan sulfate, heparin, heparansulfate, hyaluronic acid, keratan sulfate, etc.), proteoglycans (e.g.,GAGs linked to a core protein (such as via serines thereof) to formaggrecan, agrin, brevican, collagen type XVIII, leprecan, neurocan,perlecan, small leucine-rich proteoglycans, versican, or the like),fibrous proteins (e.g., collagen, elastin, fibronectin, laminin, etc.),and/or the like. Protease recognition sites (e.g., for a scaffoldmetalloproteinase (MMP)) may be incorporated into the hydrogel/matrix toallow degradation/remodeling by cells. The frequency of such sites,along with the sequence of each site may be selected to permit asuitable amount of degradation/remodeling. One or more growth factorsmay be included in the matrix when formed, or may be introduced in aliquid medium after formation. Exemplary growth factors that may besuitable include angiopoietin, bone morphogenetic proteins (BMPs),ciliary neurotropic factor, colony stimulating factors, ephrins,epidermal growth factor, erythropoietin, fibroblast growth factors,glial-derived neurotrophic factor, hepatocyte growth factor, insulin,insulin-like growth factors, interleukins, leukemia inhibitory factor,keratinocyte growth factor, neuregulins, neurotrophins, platelet-derivedgrowth factor, transforming growth factors, tumor necrosis factor(alpha), vascular endothelial growth factor, and/or the like.

Any suitable cells may populate the scaffold initially. These cells mayinclude stem cells (e.g., pluripotent stem cells), support cells, and/orthe like. The cells may be deposited in a scaffold by any suitabletechnique including bioink droplet printing, micro-contact printing,photolithography, dip pen nanolithography, and/or pipetting, amongothers.

The vessel may provide a plurality of reservoirs that are in fluidcommunication with the chamber via channels, which may be formed inshared wall(s) between the reservoirs and the chamber. Thisconfiguration may be described as a standard feeding interface. In someembodiments, 3D printing provides connection of the standard feedinginterface inside the vessel to any suitable printed structure to enablethe growth of different types of organoids.

The printed 3D structure can provide temporary scaffolding for cells ofthe appropriate type(s) as they develop into an organoid. The cells mayself-organize and produce their own extracellular matrix, which mayreplace some or all of the scaffolding.

The same may be true for internal feeding—the vessel may provide ageneral interface, which optionally may be modified by 3D printing, andthe cells may organize to best use this modified interface.

The scaffold (with or without cells) may be disposed in the receptacleof the vessel body, and the chamber may be formed from the receptacle,while the vessel is upside down. Once these processes are completed, thevessel may be turned right-side up (to its organoid-culturingorientation), and at least one reservoir overlying the chamber may befilled with feeding liquid. If there are not yet any cells inside thescaffold, suitable cells may be placed into the feeding liquid andintroduced into the scaffold together with the feeding liquid from anoverlying reservoir (or cells may be introduced via an access tube(e.g., see Example 7)).

The forming organoid may need an initial incubation time before aspecific feeding protocol can be started. The feeding protocol mayinvolve loading reservoirs with suitable media and removing media fromthe reservoirs according to a predefined schedule and/or based on thedevelopmental stage or condition of the organoid. The feeding protocolmay depend on the shape of the scaffold as well the type of organoidthat is to be formed.

The vessel may be structured to enable organoid monitoring via a bottomwindow of the chamber, which may be provided by the sealing member. Insome embodiments, the organoid may be monitored while remaining insidean incubator. Accordingly, the incubator may include an imaging systemfor organoid monitoring.

The vessel may enable light-sheet 3D imaging. The chamber of the vesselmay have two, three, or more optical windows, and light may propagateinto and/or out of the chamber via each window. For example, the vesselmay have a bottom window and one or more lateral windows, each of whichmay be planar. In some embodiments, the vessel may have a pair oflateral windows arranged opposite one another.

A plurality of vessel bodies (and vessels) may be organized as a strip.The strip may be created by pre-attaching vessel bodies to one anotherin a linear or two-dimensional array during manufacture (e.g., bybonding or forming the vessel bodies integrally with one another, suchas by injection molding). Alternatively, the strip may be created duringmanufacture or by the user by assembling individual vessel bodies with asuitable strip holder. In some embodiments, the strip holder may beconfigured to hold only one strip or two strips to allow room forimaging objectives. A strip of vessel bodies may be loaded into a 3Dprinter, to allow the printer to print a scaffold in and/or add asealing member to each of the vessel bodies.

The present disclosure enables generation of large functional organoidsby feeding the organoids with different media inside and outside. Thelarge organoids may be greater than about 0.1, 0.2, 0.5, 1, or 2millimeters, among others, in average diameter or maximum diameter.Working with large organoids is still challenging and researchers arefacing two major limitations. First, each type of organoid needsdifferent culture conditions like specific hydrogels, matrices as asubstrate, or even mechanical properties like shear force by media flow.Second, microscopy of large organoids is very challenging.State-of-the-art methods are still thin-sectioning of the organoidmaterial, staining, and image acquisition of fixed samples usingconfocal point scanning microscopes or even slide readers.

The present disclosure offers systems and methods to facilitateovercoming one or both obstacles. By using a combination of 3D printing(scaffold and/or cells) together with media exchange by gravity flow,the user may generate a unique 3D environment that is optimized for eachtype of organoid. A wide range of different organoid types may be grown.Feeding and waste removal may be addressed by fluid communicationbetween the vessel's chamber and overlying reservoirs. Integratingoptical windows into each vessel, at least one for entry of excitationlight, and another for exit of emitted light, allows monitoring livingcells of the organoid by light-sheet microscopy. Alternatively, or inaddition, the organoid may be imaged by classical widefield microscopyvia one or more of the windows. Thus, the vessel disclosed herein mayenable performance of live cell microscopy of a developing and/ordeveloped organoid. High-content and/or high-throughput microscopy maybe performed on organoids.

Further aspects of the present disclosure are described in the followingsections: (I) vessel for organoid formation, culture, monitoring, and/oranalysis, (II) methods of organoid formation, culture, monitoring,and/or analysis, and (III) examples.

I. VESSEL FOR ORGANOID FORMATION, CULTURE, MONITORING, AND/OR ANALYSIS

This section describes an exemplary vessel 50 for formation, culture,monitoring, and/or analysis of an organoid 52 (or other organizedmulti-cellular structure); see FIGS. 1 and 2 . Vessel 50 is shownschematically here, with front and back walls not visible, todistinguish top and bottom sides of the vessel that are closed (FIG. 1 )or open (FIG. 2 ).

FIG. 1 shows vessel 50 containing organoid 52 in a chamber 54, with theorganoid submerged in a medium 56 (e.g., a liquid or semi-solid culturemedium to encourage organoid growth and development). Organoid 52 may beattached to an upper region of chamber 54, such as a top wall 58thereof. (The top wall interchangeably is called a ceiling.) Lateralwalls 60 and a bottom wall 62 of the chamber may provide barriers topassage fluid, thereby rendering all of the chamber below top wall 58fluid-tight.

Vessel 50 defines a plurality of compartments 63 located over chamber54. Each compartment 63 may share a wall with chamber 54 and may or maynot be in fluid communication initially with the chamber. Exemplarycompartments include reservoirs and access tubes (see Example 7). Here,vessel 50 includes (at least) two reservoirs 64 a, 64 b for holding arespective liquid medium 66 a, 66 b over chamber 54. Each reservoir 64a, 64 b may be in fluid communication with chamber 54 independently ofeach other reservoir, via a corresponding, dedicated channel 68 a, 68 b.Each channel 68 a, 68 b may extend through top wall 58 from one ofreservoirs 64 a, 64 b to chamber 54, and may be flush with the top andbottom sides of top wall 58, or may project from the top side and/orbottom side of top wall 58 as the lumen of an annular protrusion (e.g.,see Example 4). In other examples, vessel 50 may have at least orexactly 3, 4, 5, 6, or more overlying reservoirs, each of which may bein fluid communication with chamber 54 through top wall 58, and/orindependently of each other reservoir (e.g., see Example 3).

The two or more reservoirs of vessel 50 may hold any suitable substancesto be supplied to chamber 54. Exemplary substances include nutrients,effectors, and reagents, among others. Suitable nutrients include anysubstances to facilitate the health and proliferation of cells, and thusgrowth and development of organoid 52, inside chamber 54. Exemplarynutrients may include sugars (such as glucose), amino acids, proteins,nucleotides, vitamins, minerals, fatty acids, etc. Effectors include anymolecules (such as inducers or repressors) that activate, control, orinactivate a process or action (such as differentiation, proteinsynthesis, migration, etc.). Exemplary effectors include anti-cancercompounds, growth factors, differentiation factors, oligonucleotides,mRNAs, or the like. Reagents include any compounds that facilitateanalysis of an organoid. Exemplary reagents include labels, fixationagents, and clearing agents, among others. The labels may include dyes(e.g., visible stains and/or photoluminescent dyes). Photoluminescentdyes are any substances that emit light in response to irradiation withexcitation light.

Each reservoir 64 a, 64 b may have lateral walls 70 to contain fluidlaterally. At least one lateral wall may be shared between at least oneadjacent pair of the reservoirs, as depicted in FIG. 1 . In otherexamples, at least one pair of reservoirs, of the same or differentvessels, may be in fluid communication with one another laterally,independent of chamber(s) 54 (e.g., see Example 2), such as via achannel formed in a wall 70 shared between reservoirs. Each reservoir 64a, 64 b may have an open top 72 to facilitate introduction and removalof fluid with a fluid-transfer device (e.g., a pipet or other pump).Vessel 50 may include a single, removable lid 74 that fits over thevessel to cover open top 72 of each reservoir 64 a, 64 b duringincubation in an incubator. Alternatively, the vessel may include two ormore lids that are removable independently from one another and thatcollectively cover all of the reservoirs. Each lid 74 may have a flange76 configured to vertically overlap the upper end of each reservoir andrestrict lateral motion of the lid when covering the reservoir(s),optionally without creating a tight fit. In some embodiments, the lidmay be cap that forms a fluid-tight seal at the top of one or morereservoirs (e.g., see Example 8).

FIG. 2 shows an exploded view of vessel 50 of FIG. 1 with the vesselempty. Vessel 50 may include lid 74, a vessel body 78, and a sealingmember 80. Vessel body 78 may define reservoirs 64 a, 64 b and areceptacle 82 under the reservoirs. Receptacle 82 provides top wall 58and lateral walls 60 of chamber 54. However, receptacle 82 may have anopen bottom side 84 that can be sealed with a flat sealing member 80 tocreate chamber 54, after a scaffold 86 to support organoid formation hasbeen disposed in receptacle 82 (see FIG. 1 and Section II). The sealingmember can be bonded to a bottom end of receptacle 82 (and/or vesselbody 78) to create chamber 54. In other embodiments, a sealing membercan be formed in situ to seal bottom side 84 of receptacle 82 (seeSection II).

Vessel 50 may have one or more optical windows to facilitate imaging(see FIG. 1 ). Each optical window may be used to propagate light intothe chamber, to illuminate at least a portion of chamber 54, or toreceive light from the chamber, such as for imaging. In exemplaryembodiments, vessel 50 has a bottom window 88 provided by sealing member80, and one or more lateral windows 90 provided by vessel body 78. Eachoptical window may be transparent and/or planar. Bottom window 88 may betransverse (e.g., orthogonal) to each lateral window 90, and the lateralwindows may be formed by opposite lateral walls 60 of chamber 54. (Theterm “light,” as used herein, means optical radiation, includingultraviolet radiation, visible radiation (i.e., visible light), and/orinfrared radiation.)

The components of vessel 50 may be formed of any suitable material byany suitable procedures. In exemplary embodiments, vessel body 78 may beformed of polymer, such as transparent polymer. The vessel body may haveno removable/moving parts and/or may be formed as a single piece, suchas by injection molding, such that all of the structures (e.g.,compartments) of the vessel body are formed integrally with one another.Accordingly, receptacle 82 (and/or chamber 54) and reservoirs 64 a, 64 bmay have fixed positions relative to one another and/or may benonremovably/firmly attached to one another. Sealing member 80 may beformed of glass or polymer, among others, and may be pre-formed orformed in situ, at least partially or completely inside receptacle 82.

Chamber 54 (and/or receptacle 82) and each reservoir 64 a, 64 b may haveany suitable size. Chamber 54 may have a volume of at least about 0.1,0.2, 0.3, 0.4, 0.5, 0.7, or 1 mL. The chamber may be sized to contain anorganoid of any suitable size (e.g., maximum size), such as an organoidhaving a diameter of at least about 0.2, 0.5, 1, 2, 3, 4, or 5 mm, amongothers. In exemplary embodiments, each overlying reservoir of the vesselis at least as large as, or about 2, 5, or 10 times the volume ofchamber 54 (and/or receptacle 82), such as at least about 0.5, 1, 2, 4,or 6 mL, among others. Vessel 50, and/or any of the compartmentsthereof, may have any suitable geometry. For example, each of receptacle82, chamber 54, and/or each reservoir (e.g., reservoirs 64 a, 64 b)independently may have a polygonal (e.g., rectangular), elliptical(e.g., circular), ovoid, rosette, or other shape in cross-section (e.g.,in a horizontal plane).

Accordingly, receptacle 82, chamber 54, and/or each reservoirindependently may be cylindrical, frustoconical, a rectangular prism, atapered prism, or a combination thereof, among others.

Further aspects of vessel 50 that may be suitable for organoidformation, culture, monitoring, and/or analysis are described below.

II. Methods of Organoid Formation, Culture, Monitoring, and/or Analysis

This section describes methods of forming, culturing, monitoring, and/oranalyzing organoids (or other organized multi-cellular structures) inthe vessels of the present disclosure; see FIGS. 3-15 . The method stepsdescribed in the section may be performed in any suitable order andcombination, using any of the vessels, vessel features, and/orprocedures described elsewhere herein.

FIGS. 3-10 illustrate exemplary configurations of vessel 50, organoid52, and media 56, 66 a, 66 b produced by performance of a method oforganoid culture and analysis. At least one vessel body 78 of at leastone vessel 50 may be selected for use. In some embodiments, a linear ortwo-dimensional array of vessel bodies 78 may be selected (e.g., seeExamples 1 and 6), or the array may be created after at least a subsetof the steps below have been performed.

FIG. 3 shows a vessel body 78 that has been selected and placed in aninverted orientation in which receptacle 82 is open on top. In otherwords, top wall 58 is positioned under open bottom side 84. Thisorientation takes advantage of gravity to encourage delivery of fluidonto top wall 58, while lateral walls 60 prevent lateral flow of fluidout of receptacle 82.

A scaffold 86 to be used for organoid formation may be disposed insidereceptacle 82. The scaffold may be included in a hydrogel 92 that iscreated at least partially in situ, as shown in FIGS. 4 and 5 . Forexample, scaffold 86 of hydrogel 92 may be printed in three-dimensionsby a 3D printer 94 onto top wall 58. 3D printer 94 may utilize anysuitable technology to deliver components of the scaffold. In someembodiments, the 3D printer may utilize inkjet technology, to deliverdroplets of fluid (such as droplet 95), which may contain scaffoldstructural components, cells 96, tubes, caged effectors, growth factors,etc. Alternatively, or in addition, 3D printer 94 may utilizelaser-induced forward transfer to deliver components (e.g., cells 96),among others. In other embodiments, at least part of scaffold 86 may becreated separately from vessel body 78 and then placed into receptacle82 (e.g., attached to top wall 58).

Hydrogel 92 may or may not be a thermoreversible gel. The hydrogel mayhave a gel point (a gelling temperature) above the culture temperaturefor the organoid, such that the scaffold of the hydrogel forms throughcooling as the scaffold is being printed. Alternatively, or in addition,polymerization to form the hydrogel may be photo-induced with opticalradiation, such as ultraviolet light or visible light, among others.

Scaffold 86 (and/or hydrogel 92) may horizontally overlap the bottom endof one or more channels 68 a, 68 b. For example, in the depictedembodiment, a through-axis (e.g., a vertical axis) defined by channel 68b intersects hydrogel 92 and extends through scaffold 86. In otherembodiments, scaffold 86 (and/or hydrogel 92) may overlap a plurality ofchannels 68 a, 68 b.

One or more channels 68 a, 68 b may be extended by 3D printing duringcreation of scaffold 86 in hydrogel 92, to facilitate supplyingnutrients and/or effectors to hydrogel 92 and cells 96 therein duringorganoid formation and culture. The resulting channel extensions may beone or more laterally-permeable tubes that are embedded in hydrogel 92,and may be branched to form a channel network inside the hydrogel. Thechannel network and/or a laterally-permeable tube embedded in hydrogel92 may extend between at least a pair of channels 68 a, 68 b of vesselbody 78. Effectors (such as differentiation factors) may be supplied viathe channel network to establish concentration gradients inside thehydrogel for more controlled stem cell differentiation and organoidformation. The tubes may be created by photo-induced or thermalpolymerization, among others. Pre-formed tubes also or alternatively maybe incorporated into the hydrogel or scaffold for additionalfacilitation of liquid flow within the hydrogel/scaffold.

An open side of receptacle 82 may be sealed hermetically to produce achamber 54 containing hydrogel 92 and scaffold 86 therein. A sealingmember 80 may be bonded to the end of receptacle 82, as shown in FIG. 6. Alternatively, as described below, sealing member 80 may be created insitu by polymerization and/or solidification of a sealing fluid, atleast partially inside receptacle 82. Receptacle 82 may be sealed beforeor after cells 96 have been placed into the receptacle, and/or before orafter a medium 56 has been into receptacle 82 (and/or chamber 54) aroundhydrogel 92.

Media 56, 66 a, 66 b may be disposed in chamber 54 and each reservoir 64a, 64 b of vessel 50; see FIG. 7 . The same medium may be introducedinto each chamber and reservoir, or different media may be introduced,which may create a concentration gradient of one or more nutrientsand/or effectors. As explained above, medium 56 may be introducedinitially via an open side of receptacle 82, before the receptacle issealed to create chamber 54. To prevent leakage of medium 56 fromreceptacle 82 via one or more channels 68 a, 68 b before chamber 54 iscreated, an end of each channel may be covered with hydrogel 92 (or adifferent hydrogel). The different hydrogel may be configured to meltbelow the incubation temperature (such as 37° C.) for organoid formationand culture. Alternatively, medium 56 may be introduced into chamber 54(after the chamber is formed) from at least one reservoir 64 a, 64 b viaat least one channel 68 a, 68 b.

Gravity may drive fluid into chamber 54 from one of the reservoirs,and/or between the reservoirs via the chamber if the fluid levels in thereservoirs are different. For example, in FIG. 7 , the top of medium 66a in reservoir 64 a is higher than the top of medium 66 b in reservoir64 b. Accordingly, gravity drives flow of medium 66 a into chamber 54via channel 68 a (which functions as an inlet), and out of chamber 54via channel 68 b (which functions as an outlet). Over time, the levelsof media 66 a, 66 b will tend to equalize. Accordingly, fluid may beadded to and/or removed from one or more of the reservoirs to drivefurther fluid flow in the same or the reverse direction, or theverticality of vessel 50 may be changed periodically (e.g., see Example2).

FIGS. 8 and 9 show vessel 50 containing organoid 52. The organoid isformed by proliferation and differentiation of cells 96 in hydrogel 92.In FIG. 9 , the size and shape of organoid 52 have changed relative toFIG. 8 , as scaffold 86 is remodeled by cells of the organoid. In otherembodiments, the final size and shape of organoid 52 may be definedsubstantially by the size and shape of scaffold 86 when formed.

FIG. 10 shows an exemplary imaging system 100 for capturing an image 102of organoid 52 by light-sheet microscopy. Imaging system may include anillumination assembly 104 including a light source 106 to generate lightfor a light sheet 108. The light sheet may be oriented horizontally, asdepicted in FIG. 10 , and may propagate through organoid 52 from one oflateral windows 90. Alternatively, light sheet 108 may be orientedvertically, and may propagate through organoid vertically from bottomwindow 88. Light may be collected from organoid 52 with an objective 110for capture by an image sensor 112. Light that propagates through bottomwindow 88 may be collected, as in the depicted embodiment, or light maybe collected from one of lateral windows 90. Light sheet 108 may bemoved, by corresponding motion of illumination assembly 104 (or vessel50), to permit capture of a stack of images, thereby providingthree-dimensional image data for the organoid. If organoid 52 no longerneeds to be viable, the organoid may be fixed and cleared beforeimaging, by supplying fixing and clearing reagents from one or more ofreservoirs 64 a, 64 b through top wall 58 of chamber 54.

FIGS. 11-13 illustrate an alternative, in situ approach to sealing openside 84 of receptacle 82 to produce chamber 54. FIG. 11 shows vesselbody 78 inverted, after formation of hydrogel 92 including scaffold 86.A filler hydrogel 113 also has been dispensed into open receptacle 82via open side 84, which may prevent leakage from receptacle 82 throughchannel 68 a. The filler hydrogel may be configured to melt or dissolveonce organoid culture begins. FIG. 12 shows 3D printer 94 dispensing asealing liquid 114 into receptacle 82. FIG. 13 shows sealing liquid 114solidifying to hermetically seal the open side of receptacle 82 tocreate chamber 54. The sealing liquid may be configured to be solidifiedto produce a thermoplastic polymer or a thermosetting polymer, amongothers. Exemplary thermoplastic polymers include a thermoplasticelastomer or wax, preferably having a melting point of less than 100° C.A lower melting point may be desirable, but at least about 50° C. Thethermoplastic polymer may be dispensed in liquid form onto the surfaceof filler hydrogel 113, at a temperature above the melting temperatureof the polymer, to form a sealing layer 116, which hardens as the layercools to create a sealing member 80.

In other examples, the layer may be solidifiable to create athermosetting polymer. FIG. 13 shows an example in which sealing layer116 is solidified to a thermosetting polymer by irradiation with opticalradiation (e.g., ultraviolet light) from illumination assembly 104. Theillumination assembly forms a light sheet 108 that preferentiallyirradiates sealing layer 116 with ultraviolet light, thereby minimizingphotodamage to scaffold 86 and/or cells 96 already present in receptacle82. FIG. 14 shows vessel body 78 flipped over to its organoid-culturingorientation, with sealing layer 116 solidified to form sealing member80, and media 56, 66 a, 66 b in chamber 54 and reservoirs 64 a, 64 b.Objective 110 of an imaging system is collecting light from chamber 54to form an image. Medium 56 and sealing layer 116 may have substantiallythe same refractive index, to improve image quality. FIG. 15 illustratesa modified in situ approach for sealing an open side of receptacle 82 ofvessel body 78 (compare with FIG. 13 ). A radiation-blocking layer 118may be formed on the surface of filler hydrogel 113, before layer 116 ofa thermosetting sealing liquid is added to receptacle 82. Layer 116 maybe irradiated with optical radiation from a light source 120 positionedabove vessel body 78, to encourage solidification of layer 116 to form asealing member 80. Radiation-blocking layer 118 shields hydrogel 92 fromphotodamage.

III. EXAMPLES

This section describes further embodiments of systems and methods fororganoid formation, culture, monitoring, and/or analysis. Theseembodiments are intended for illustration only and should not limit theentire scope of the present disclosure.

Example 1 Strip for Organoid Cultures

This example describes an exemplary strip 130 for forming, culturing,monitoring, and/or analyzing an array of organoids; see FIG. 16 .

Strip 130 may include an array of vessel bodies 78 connected to oneanother. The vessel bodies may be formed integrally with one another, orformed separately and then connected to one another after formation(and/or after receptacles 82 are sealed to form chambers 54 (also seeFIG. 1 ). Separately-formed vessel bodies 78 may be connected to oneanother by bonding, an interference fit, fasteners, or a holder (e.g.,see Example 6), among others. The vessel bodies may be connected to oneanother to create a linear or at least two-dimensional array ofreceptacles 82.

Example 2 Fluid Transfer Between Vessel Reservoirs

This example describes exemplary configurations to transfer fluidbetween reservoirs 64 a, 64 b of the same or different vessels 50; seeFIG. 17, 17A, 17B, and 18 .

FIG. 17 shows strip 130 during culture of organoids 52 in chambers 54.Each vessel 50 has a separate flow cycle indicated by arrows 132, 134.Flow 132 through each chamber 54, between channels 68 b and 68 a, isdriven by gravity as a result of the difference in levels of media 66 a,66 b in respective reservoirs 64 a, 64 b. Replenishing flow 134 fromreservoir 64 a to reservoir 64 b is driven by a pump 136. The flow rateof pump 136 can be adjusted to keep the respective levels of media 66 a,66 b substantially constant, such that fluid passes through chamber 54at a substantially constant rate. This flow may apply pressure onorganoid 52, which can promote its growth and development. In otherexamples, the flow rate of pump 136 can be varied, to apply a varyingpressure on organoid 52.

In other embodiments, pump 136 can be eliminated, as shown for vesselstrip 130 in FIGS. 17A and 17B. Strip 130 may be rocked to tilt theholder from vertical at a suitable rate, in order to periodicallyreverse the direction of gravity-driven flow 132. In other words, theholder may be tilted in one direction to drive fluid from reservoir 64 ato 64 b within each vessel 50, and then tilted in the oppositerotational direction to drive fluid from reservoir 64 b to 64 a withineach vessel 50, via channels 68 a, 68 b.

FIG. 18 shows another strip 140 during culture of organoids 52 inchambers 54. Strip 140 is similar to strip 130 of FIGS. 16 and 17 , withflow 132 driven between channels 68 a, 68 b of each vessel 50. However,a respective channel 142 provides fluid communication between eachadjacent pair of vessels 50. More specifically, each channel 142 allowsfluid to flow directly from reservoir 64 a of one vessel 50 to reservoir64 b of an adjacent vessel 50, as indicated by flow arrow 144, or viceversa. To maintain fluid circulation, strip 140 can be periodicallytilted (as in FIG. 18 ) to drive fluid from left to right, toward oneend of the holder, and then tilted in the opposite rotational direction,to drive fluid from right to left toward the opposite end of the holder.A pump 146 may transfer media between reservoirs 64 a, 64 b located atopposite ends of strip 140, indicated by a flow arrow at 148. The pumpcan be used instead of, or in addition to, periodically tilting strip140.

Example 3 Vessel Embodiment

This example describes an exemplary embodiment 150 of vessel 50 ofSection I having a vessel body 78 formed by injection molding anddefining four reservoirs 64 a-64 d; see FIGS. 19-25 . Each reservoir 64a-64 d may be in fluid communication with receptacle 82 (and/or chamber54) via a corresponding channel 68 a-68 d extending through top wall 58of receptacle 82 (and/or chamber 54) (see FIGS. 23 and 25 ). The upperend of each channel may be flush with top wall 58 (see FIG. 25 ).

Various optical windows may be incorporated into vessel 150. A planarsealing member 80 (e.g., similar to a microscope cover slip) may providea bottom window 88 of chamber 54, after the sealing member has beenbonded to a bottom end surface 152 of vessel body 78 (see FIG. 21 ). Thevessel body may include a pair of lateral windows 90 (see FIGS. 19-21 ),which may be slanted somewhat from vertical to facilitate manufacture byinjection molding (see FIG. 24 ).

Example 4 Vessels with Tubes

This example describes exemplary vessels having tubes that arepre-formed and/or formed in situ; see FIGS. 26 and 27 .

FIG. 26 shows another embodiment 160 of vessel 50, for comparison withvessel 150 in FIG. 25 . Channels 68 a-68 d of vessel 160 differ fromthose of vessel 150. More particularly, channels 68 b and 68 c aredefined in part by annular protrusions 162 projecting from a bottom sideof top wall 58 of receptacle 82 (and/or chamber 54). The lumens of anyor all of the channels may be formed at least in part by an annularprotrusion.

FIG. 27 shows a laterally porous, tubular extension 164 that may beformed in situ on one or more annular protrusions 162 by 3D printing.Each tubular extension may or may not be branched. In the depictedembodiment, tubular extension 164 connects channels 68 b and 68 c to oneanother. In other embodiments, a porous, tubular network may be formed,which may connect any suitable subset or all of the channels to oneanother. Each tubular extension 164 may be supported by, and/or embeddedin hydrogel 92 (see Section II). The tubular extension may have the samecomposition as the hydrogel or may have a different composition, to, forexample, minimize/promote cell attachment and/or remodeling, amongothers.

Example 5 Vessel with Electromagnet

This example describes an exemplary embodiment 170 of vessel 50 havingan electromagnet 172 positioned to attract ferromagnetically-labeledcells 174 when the electromagnet is energized (i.e., turned on); seeFIGS. 28-30 .

Vessel 170 may be constructed similarly to vessel 150 and may have anysuitable combination of vessel features disclosed herein (see FIG. 28 ).Electromagnet 172 may have a working end 176 located very near or inreceptacle 82 and/or chamber 54, close to at least one of channels 64a-64 d. In the depicted embodiment, working end 176 is centered betweenchannels 64 b and 64 c. The electromagnet may be energized via an upperend 178 thereof.

FIGS. 29 and 30 illustrate how electromagnet 172 may be utilized toattract ferromagnetically-labeled cells 174 to hydrogel 92 and/orscaffold 86 inside chamber 54. Scaffold 86 and hydrogel 92 may be formedas described above. Ferromagnetically-labeled cells 174 may beintroduced into receptacle with medium 56, before the receptacle issealed to form chamber 54, or after sealing, from one or more overlyingreservoirs via one or more channels 68 a-68 d. In any event,ferromagnetically-labeled cells 174 are initially outside of hydrogel92, and may settle to the bottom chamber 54, as in FIG. 29 , before theelectromagnet is turned on. FIG. 30 shows how energizing theelectromagnet attracts ferromagnetically-labeled cells 174 to and/orinto scaffold 86.

Ferromagnetically-labeled cells may be associated with a ferromagneticmaterial (e.g., compounds containing iron, cobalt, and/or nickel, amongothers) before the cells are introduced into the receptacle/chamber. Forexample, cells may be fed ferromagnetic particles (such as iron oxideparticles) to render the cells ferromagnetic or may coated with aferromagnetic probe, among others.

Example 6 Rack for Vessel Modules

This example describes exemplary racks 180, 182 to hold vessels 150,respectively in a linear array or two-dimensional array to form a strip130; see FIGS. 31-34 .

Each rack 180, 182 (interchangeably called a holder) has a series ofopenings 184 to receive vessel bodies 78 of vessels 150. Openings 184may be arranged along the same line (rack 180) or in a rectangular gridpattern (rack 182), among others. Each vessel body 78 may be removablyplaced into opening 184. The opening may be sized and shaped to preventthe vessel body from passing completely through the opening. The vesselbody may be coupled to opening 184 by any suitable mechanism, includinga snap-fit or a separate retainer, among others.

Example 7 Vessels with Access Tubes

This example describes exemplary vessels that include access tubes, anduse of the access tubes to receive various instruments; see FIGS. 35-51.

FIG. 35 shows an embodiment 190 of vessel 50 including vessel body 78sealed at its bottom end with a sealing member 80 to form a chamber 54.Vessel body 78 is similar to that described above for vessel 160 (seeFIG. 26 ), except that the vessel body defines at least one access tube192. The access tube is open at its top end 194, but may have a bottomend that is closed by a barrier 196. The barrier may be a wall region oftop wall 58 of the chamber that is shared between access tube 192 andchamber 54, and that prevents fluid communication between the accesstube and the chamber, while the barrier is intact. Chamber 54 andreservoirs 64 a, 64 b are not shown as containing fluid (e.g., medium orother liquid) in any of the figures of Example 7 to simplify thedrawings.

Barrier 196 may be configured to be breached by a sharp or bluntinstrument, as described further below, to access chamber 54.Accordingly, the barrier may be thinner than adjacent regions of topwall 58, as shown in FIG. 35 , and/or may include predefined structure(e.g., predefined frangible regions of lesser thickness) at which thebarrier can be preferentially torn or otherwise breached with a suitableinstrument.

FIG. 35 shows an exemplary instrument 198 inserted into access tube 192.Here, instrument 198 is a permanent magnet 200, with a pole 202 of themagnet adjacent barrier 196. In other embodiments, the magnet can be anelectromagnet or any of the other instruments disclosed herein. Magnet200 may be sufficiently strong to form a magnetic field that extendsinto chamber 54 while barrier 196 is intact. In other cases, the bottomend of magnet 200 may be inserted into chamber 54 after barrier 196 isbreached (and even may breach the barrier). Whether or not the magnetenters chamber 54, the magnet can apply attractive force toferromagnetic items (e.g., ferromagnetically-labeled cells) insidechamber 54. The magnet may be retractable/removable and/or may be firmlyattached to access tube 192.

FIG. 36 shows vessel 190 taken with an organoid 52 present in chamber54, and with magnet 200 replaced by an instrument 198 structured as aneedle 204 (compare with FIG. 35 ). A sharp tip 206 of needle 204 haspierced barrier 196 (now referred to as breached barrier 196′) and hasentered organoid 52. The needle may be hollow or solid. In either case,the needle may permit a sample of organoid 52 to be collected andremoved from chamber 54 via access tube 192, for biopsy of the organoid.Alternatively, or in addition, the needle may be used to introducefluid, cells, and/or effectors, among others into organoid 52 andchamber 54. For example, the needle may permit tissue or cells to betransplanted to the organoid, to achieve xenografting, allografting, orhomografting.

FIG. 37 shows vessel 190 as in FIG. 36 but with instrument 198 being asensor/electrode 208 including an electrical connector 210 (e.g., one ormore electrically conductive wires). A sensing/stimulating end region212 of sensor/electrode 208 has pierced barrier 196 (now breachedbarrier 196′) and entered organoid 52. In other embodiments, barrier 196may be breached first by a different instrument 198, such as a dedicatedbreaching instrument, and then replaced by sensor/electrode 208 (or anyof the other instruments disclosed herein, among others).Sensor/electrode 208 may include at least one electrode or an array ofelectrodes for electrical stimulation of organoid 52. Alternatively, orin addition, sensor/electrode 208 may include any suitable sensor(s),such as an electrical sensor (e.g., a capacitance sensor), a pH sensorto measure pH, an electrochemical sensor, an oxygen sensor, a CO₂sensor, and/or the like.

FIGS. 38-40 show an instrument 198 structured as a breaching instrument214 before, during, and after barrier 196 at the bottom end of accesstube 192 is pierced. In FIG. 38 , a pointed tip 216 of breachinginstrument 214 is traveling downward, indicated by a motion arrow at218, and has almost reached barrier 196. In FIG. 39 , tip 216 is incontact with barrier 196 and is applying a deforming force to thebarrier. In FIG. 40 , tip 216 has passed completely through the barrierand entered chamber 54. The wall region forming barrier 196 may besufficiently elastic to maintain radial contact with breachinginstrument 214, after the barrier has been pierced. This radial contactmay form a fluid-tight seal 220 between vessel body 78 (at breachedbarrier 216′) and breaching instrument 214, which may substantiallyprevent fluid in chamber 54 from traveling upward into access tube 192.

Vessel 190 may have only one or a plurality of access tubes 192. Forexample, FIG. 41 shows vessel 190 sectioned generally as in FIG. 24 ,such that three access tubes 192 are visible. Each access tube 192 mayend at a respective barrier 196, as described above. Moreover, eachbarrier 196 may be a wall region of top wall 58 that is locatedintermediate, and/or shared between, the corresponding access tube 192and chamber 54. Accordingly, the wall region may or may not be a commonwall region. In the depicted embodiment, a respective breachinginstrument 214 is disposed in each of the three access tubes 192, withonly one of the breaching instruments extending into chamber 54. (Thebreached barrier is indicated with 196′.) In other embodiments, anysuitable number of barriers 196 may be breached to provide access tochamber 54 with any suitable combination of instruments 198.

FIGS. 42-44 show fragmentary sectional views of a different embodiment230 of vessel 50, taken as in FIGS. 38-40 , before, during, and afterbarrier 196 is breached with breaching instrument 214. Barrier 196 ofvessel 230 has a frangible web 232 at which the barrier ispreferentially torn in response to pressure applied by breachinginstrument 214. In FIG. 44 , the end of breaching instrument 214 hasentered chamber 54. Fluid may travel upward from chamber 54 into accesstube 192, or this fluid travel may be restricted by a close radial fitof breaching instrument 214 with access tube 192 and/or a hermetic sealaround the breaching instrument at the top of access tube 192, amongothers.

FIG. 45 shows vessel 190 with an Attenuated Total Reflectance (ATR)fiberoptic probe 234 disposed in access tube 192 and extending intoorganoid 52 in chamber 54. ATR probe 234 has an illumination fiber 236to direct light from a light source to a bottom end of probe 234,indicated at 238, and a sensor fiber 240, to direct light from thebottom end to an optical sensor (e.g., a spectrometer, photometer, orthe like), indicated at 242. Probe 234 may permit IR spectroscopy (e.g.,for chemical analysis of the organoid), Raman spectroscopy (such assurface-enhanced Raman spectroscopy), or the like.

FIG. 46 shows vessel 190 with a fiberoptic imaging probe 244 disposed inaccess tube 192 and extending into organoid 52 in chamber 54. Imagingprobe 244 may have an endoscope front lens 246 and a fiber bundle 248for propagation of light from front lens 246 to an image sensor. Theimaging probe may permit endoscopic imaging, confocal imaging, and/orillumination only (e.g., for imaging with an image sensor not coupled tofiber bundle 248, optogenetics, etc.), among others.

FIG. 47 shows vessel 190 with an illumination device 250 disposed inaccess tube 192 and extending into organoid 52 in chamber 54.Illumination device 250 may include an optical fiber 252 to direct lightfrom a light source 254 to an outlet aperture 256 at a distal end ofoptical fiber 252. Exemplary uses for illumination device 250 includeilluminating organoid 52 for imaging by an image sensor not coupled tooptical fiber 252, as a light source for wavefront sensing (adaptiveoptics), as a light source for optogenetics, etc.

FIG. 48 shows vessel 190 with a pneumatic device 258 located in accesstube 192. Pneumatic device 258 has an expandable balloon 260 locatedinside organoid 52. The balloon is operatively connected to a pressuremodulating device, such as a pump 262, which can inflate balloon 260 tocreate internal mechanical strain in organoid 52, indicated at 264.Pneumatic device 258 can inflate and deflate balloon 260, to expand andcontract it as needed. Balloon 260 may be printed along with scaffold86, and connected later to a tube 266 of pneumatic device 258. In otherembodiments, balloon 260 may be introduced into organoid 52 via a needleor other sharp object.

FIG. 49 shows vessel 190 as in FIG. 41 , but with a pair of pneumaticdevices 258 a, 258 b disposed in respective access tubes 192. Each ofthe pneumatic devices is similar to that of FIG. 48 except for havingexpandable balloons 260 located outside the organoid to create externalmechanical strain on opposite sides of organoid 52.

FIGS. 50 and 51 show vessel 190 viewed respectively as in FIGS. 35 and36 , but with a pair of mounting magnets 270 a, 270 b extending intochamber 54 via corresponding access tubes 192. Magnets 270 a, 270 bmagnetically attract respective magnets 272 a, 272 b, which are alreadyattached to a premanufactured scaffold structure 274 or biochip viamagnetic attraction.

Example 8 Vessel with Flexible Membrane

This example describes an exemplary vessel 190 utilizing one or moreflexible membranes (interchangeably called diaphragms) to drive fluidflow within the vessel; see FIGS. 52 and 53 .

Vessel 190 includes a cap 282 mounted on the top of vessel body 78. Cap282 forms a hermetic seal with vessel body 78. The cap includes a frame284 that fits tightly on the top edges of vessel body 78. At least oneflexible membrane 286 is mounted to frame 284 and covers at least tworeservoirs (e.g., 64 a, 64 b). An undeformed configuration of flexiblemembrane is shown dashed, at 288. Pressure 290 can be applied toflexible membrane 286 over reservoir 64 a or 64 b to push the membranedown, indicated at 292. This pressure drives fluid from one reservoir tothe other reservoir via chamber 54, indicated by arrows 294, 296.Flexible membrane 286 may deflect upward in response, indicated by 298.Pressure may be applied to flexible membrane 286 alternatively overreservoirs 64 a, 64 b, as shown in FIGS. 52 and 53 , to drive fluid inopposite directions between the reservoirs.

Example 9 Selected Embodiments

This example describes selected embodiments of the present disclosure asa series of indexed paragraphs.

Paragraph A1. A method of organoid culture and/or analysis, the methodcomprising: (a) sealing an open side of a receptacle to create achamber; and (b) forming an organoid in the chamber.

Paragraph A2. The method of paragraph A1, wherein sealing includesattaching a sealing member to the open side of the receptacle.

Paragraph A3. The method of paragraph A2, wherein attaching a sealingmember includes bonding the sealing member to the receptacle.

Paragraph A4. The method of paragraph A3, wherein bonding includesbonding a pre-made sealing member to the receptacle. Paragraph A5. Themethod of paragraph A2 or A3, wherein sealing includeshardening/solidifying a sealing material at least partially in thereceptacle to form the sealing member.

Paragraph A6. The method of paragraph A5, wherein the sealing materialincludes a thermoset resin. Paragraph A7. The method of paragraph A6,wherein sealing includes forming a layer of the thermoset resin in thereceptacle, and irradiating the layer of the thermoset resin withelectromagnetic radiation, such as ultraviolet light, to cure thethermoset resin.

Paragraph A8. The method of paragraph A7, wherein the receptaclecontains a scaffold to promote organoid formation, and whereinirradiating is performed with a sheet of light positioned and orientedto preferentially irradiate the layer of thermoset resin relative to thescaffold.

Paragraph A9. The method of paragraph A7, wherein the receptaclecontains a scaffold to promote organoid formation and also contains alight-blocking layer located intermediate the layer of the thermosetresin and the scaffold, and wherein irradiating is performed such thatlight propagates through the layer of the thermoset resin to thelight-blocking layer, which substantially shields the scaffold from thelight.

Paragraph A10. The method of any of paragraphs A7 to A9, wherein forminga layer of the thermoset resin includes depositing the thermoset resinwith a 3D printer onto a hydrogel located in the receptacle.

Paragraph A11. The method of paragraph A10, wherein the hydrogelincludes a scaffold to support organoid formation.

Paragraph Al2. The method of paragraph A11, wherein the hydrogelincludes a first hydrogel to promote organoid formation and a secondhydrogel to temporarily support the layer of the thermoset resin, andwherein the second hydrogel is configured to substantially melt ordissolve when the chamber is used for organoid culture.

Paragraph A13. The method of any of paragraphs A1 to A3 and A5, whereinthe receptacle contains a scaffold to promote organoid formation, andwherein sealing includes forming a layer of thermoplastic material overa hydrogel containing the scaffold, and hardening the layer ofthermoplastic material by cooling to hermetically seal the open side ofthe receptacle.

Paragraph A14. The method of any of paragraphs A1 to A13, wherein thereceptacle is defined by a vessel body, and wherein sealing includesattaching a sealing member to a bottom end of the vessel body.

Paragraph A15. The method of any of paragraphs A1 to A14, the methodfurther comprising introducing fluid and/or at least one substance intothe chamber from an overlying reservoir (for contact with the organoid),and wherein, optionally, the chamber and the overlying reservoir areformed integrally with one another.

Paragraph A16. The method of paragraph A15, wherein introducing includespassing the fluid and/or at least one substance into the chamber via achannel extending from the overlying reservoir to the chamber, andwherein, optionally, the channel is formed integrally with the overlyingreservoir and the chamber.

Paragraph A17. The method of paragraph A15 or A16, wherein introducingincludes passing nutrients through a top wall of the chamber to feed theorganoid.

Paragraph A18. The method of any of paragraphs A15 to A17, whereinintroducing includes passing a label through a top wall of the chamberto label at least a portion of the organoid.

Paragraph A19. The method of any of paragraphs A15 to A18, whereinintroducing includes

-   (i) passing one or more effectors through a top wall of the chamber,    and wherein the one or more effectors are selected from the group    consisting of differentiation factors, growth factors, anti-cancer    compounds, expression vectors, viruses, oligonucleotides, messenger    RNAs, and small-interfering RNAs; and/or-   (ii) passing molecules through a top wall of the chamber, wherein    the molecules are configured for genome editing of an organoid    (e.g., deletion, insertion, or substitution of one or more    nucleotides of a target sequence within cells of the organoid), such    as genome editing using a CRISPR-Cas system, a TALEN system, or the    like; and/or-   (iii) passing molecules through a top wall of the chamber to label    or one or more genomic loci of an organoid in vivo and/or to alter    expression of one or more genes by binding to those genes, wherein    the molecules are based on a CRISPR-Cas system, a TALEN system, or    the like.

The molecules of (ii) or (iii) above may include any combination of aguide RNA, an effector molecule for CRISPR-Cas expression, a virus thatcontains an expression vector for a guide RNA and/or a CRISPR-Cas systemprotein, or a CRISPR-Cas protein.

Paragraph A20. The method of any of paragraphs A15 or A19, whereinintroducing includes passing one or more fixation agents and/or clearingagents through a top wall of the chamber.

Paragraph A21. The method of any of paragraphs A15 to A20, whereinintroducing includes driving fluid into the chamber from the reservoirwith gravity.

Paragraph A22. The method of any of paragraphs A15 to A21, whereinintroducing incudes driving fluid from an inlet to an outlet of thechamber, and wherein each of the inlet and the outlet includes arespective channel that extends through a top wall of the chamber.

Paragraph A23. The method of any of paragraphs A15 to A22, wherein thechamber is in separate fluid communication with first and secondoverlying reservoirs, and wherein introducing includes introducing fluidand/or at least one substance into the chamber from each of the firstand second overlying reservoirs.

Paragraph A24. The method of paragraph A23, wherein the organoid definesan interior space located inside the organoid and an exterior spacelocated outside the organoid and within the chamber, and wherein fluidheld by the first overlying reservoir is supplied to the interior spaceand fluid held by the second overlying reservoir is supplied to theexterior space.

Paragraph A25. The method of any of paragraphs A15 to A24, wherein thechamber is in separate fluid communication with first and secondreservoirs, the method further comprising transferring fluid from thesecond reservoir to the first reservoir with a pump to promotegravity-driven flow from the first reservoir to the second reservoir viathe chamber.

Paragraph A26. The method of paragraph A25, wherein the pump transfersfluid from the second reservoir to the first reservoir at a rate thatsubstantially matches a rate of the gravity-driven flow.

Paragraph A27. The method of paragraph A26, wherein the chamber is inseparate fluid communication with first and second reservoirs defined bya vessel body, the method further comprising tilting the vessel bodyback and forth to alternately produce gravity-driven flow from the firstreservoir to the second reservoir and from the second reservoir to thefirst reservoir.

Paragraph A28. The method of any of paragraphs A15 to A27, wherein firstand second reservoirs are in fluid communication with the chamber viarespective first and second channels, wherein the first and secondreservoirs are in direct fluid communication with one another via athird channel that is above and spaced from a top wall of the chamber.

Paragraph A29. The method of any of paragraphs A15 to A28, wherein afirst reservoir and a second reservoir overlie the chamber and are inseparate fluid communication with the chamber, the method furthercomprising disposing a flexible membrane over the first reservoir, andapplying pressure to a top side of the flexible membrane to drive fluidfrom the first reservoir to the second reservoir via the chamber.

Paragraph A30. The method of paragraph A29, wherein disposing a flexiblemembrane includes disposing a flexible membrane over each of the firstand second reservoirs, and wherein applying pressure includesalternately applying pressure to the top side of the flexible membraneover the first reservoir and to the top side of the flexible membraneover the second reservoir, to alternatively drive fluid between thefirst and second reservoirs in opposite directions.

Paragraph A31. The method of any of paragraphs A1 to A30, furthercomprising disposing a scaffold inside the receptacle before the openside of the receptacle is sealed, the scaffold being configured topromote organoid formation. Paragraph A32. The method of paragraph A31,wherein disposing a scaffold includes creating the scaffold inside thereceptacle.

Paragraph A33. The method of paragraph A31, wherein disposing a scaffoldincludes placing a pre-made scaffold into the receptacle.

Paragraph A34. The method of any of paragraphs A31 to A33, furthercomprising introducing biological cells into the receptacle before theopen side of the receptacle is sealed.

Paragraph A35. The method of paragraph A34, wherein the biological cellsinclude stem cells.

Paragraph A36. The method of paragraph A34 or A35, wherein thebiological cells are introduced into the scaffold as the scaffold isbeing created.

Paragraph A37. The method of any of paragraphs A32 and A34 to A36,wherein creating a scaffold is performed by 3D printing.

Paragraph A38. The method of paragraph A37, wherein creating a scaffoldincludes 3D printing at least two different hydrogels in the receptacle.

Paragraph A39. The method of paragraph A38, wherein at least one of thehydrogels contains cells once 3D-printed, to realize anarbitrarily-shaped 3D scaffold promoting the formation of a specificorganoid.

Paragraph A40. The method of any of paragraphs A31 to A39, wherein thereceptacle has a wall opposite the open side, and wherein the scaffoldis attached to the wall.

Paragraph A41. The method of any of paragraphs A31 to A40, furthercomprising introducing biological cells for forming the organoid intothe chamber after the open side of the receptacle is sealed to createthe chamber.

Paragraph A42. The method of any of paragraphs A31 to A41, wherein thechamber has a top wall, and wherein the organoid is supported by the topwall when formed.

Paragraph A43. The method of any of paragraphs A31 to A42, wherein thechamber is in fluid communication with a plurality of overlyingreservoirs via channels, the method further comprising creating anextension of one or more of the channels by 3D printing, and,optionally, embedding the extension in a hydrogel.

Paragraph A44. The method of paragraph A43, further comprisingelongating and branching each extension by 3D printing to create one ormore laterally permeable tubes for supplying substances to cells insidethe hydrogel.

Paragraph A45. The method of paragraph A44, further comprising supplyingone or more effectors via the one or more laterally permeable tubes toestablish one or more concentration gradients of the effectors insidethe chamber for more controlled stem cell differentiation and organoidformation.

Paragraph A46. The method of any of paragraphs A43 to A45, furthercomprising incorporating polymer or metal microtubes into the hydrogelto facilitate fluid flow.

Paragraph A47. The method of any of paragraphs A1 to A46, the methodfurther comprising collecting data related to the organoid while theorganoid remains in the chamber.

Paragraph A48. The method of paragraph A47, wherein collecting dataincludes capturing an image of at least a portion of the organoid.

Paragraph A49. The method of paragraph A48, wherein capturing includescapturing an image by light-sheet microscopy.

Paragraph A50. The method of paragraph A48 or A49, wherein capturingincludes detecting photoluminescence from the organoid.

Paragraph A51. The method of any of paragraphs A48 to A50, whereincapturing includes capturing a stack of images representing a 3Dstructure of at least a portion of the organoid.

Paragraph A52. The method of paragraph A51, wherein capturing includesilluminating at least a portion of the organoid via a lateral window ofthe chamber, and detecting optical radiation that has propagated out ofthe chamber via a bottom window of the chamber, or vice versa.

Paragraph A53. The method of any of paragraphs A47 to A52, whereincollecting data includes assaying fluid from the chamber or an overlyingcompartment for an analyte.

Paragraph A54. The method of any of paragraphs A47 to A53, whereincollecting data is performed with a sensor located at least partiallyinside the chamber.

Paragraph A55. The method of any of paragraphs A47 to A54, whereincollecting data includes collecting data from cells and/or fluid removedfrom the chamber while the bottom thereof remains sealed.

Paragraph A56. The method of paragraph A55, wherein collecting dataincludes forming an opening in a top wall of the chamber and removingcells and/or fluid from the chamber via the opening.

Paragraph A57. The method of any of paragraphs Al to A56, the methodfurther comprising creating an opening in a wall of the chamber; andinserting an end of an instrument into the chamber from the opening.

Paragraph A58. The method of paragraph A57, wherein a plurality ofoverlying compartments share a common wall with the chamber, and whereinthe opening is created in the common wall at a bottom end of one of theoverlying compartments.

Paragraph A59. The method of paragraph A58, wherein the plurality ofoverlying compartments includes a plurality of reservoirs and one ormore access tubes, and wherein the opening is created at the bottom endof one of the access tubes.

Paragraph A60. The method of paragraph A58 or A59, wherein the openingis created with the instrument, and wherein creating an opening includesbreaching the common wall with the end of the instrument.

Paragraph A61. The method of any of paragraphs A57 to A60, wherein theend of the instrument is a sharp end.

Paragraph A62. The method of paragraph A60 or A61, wherein creating anopening includes forming a fluid-tight seal between the instrument andthe common wall at the opening.

Paragraph A63. The method of any of paragraphs A60 to A62, wherein thecommon wall defines a feature at which the common wall is configured tobe torn by mechanical pressure exerted on the common wall via theinstrument.

Paragraph A64. The method of any of paragraphs A57 to A63, wherein theinstrument is selected from the group consisting of a needle, a lightguide operatively connected to a light source, an endoscope, anelectrode, an ATR probe, a source of pneumatic/hydraulic pressureoptionally coupled to a balloon, and a magnet.

Paragraph A65. The method of any of paragraphs A57 to A64, wherein theinstrument includes a sensor located at the end thereof.

Paragraph A66. The method of paragraph A65, further comprising sensing aparameter of the chamber and/or organoid using the sensor.

Paragraph A67. The method of any of paragraphs A57 to A66, wherein theinstrument includes an electrode configured to electrically stimulatethe organoid.

Paragraph A68. The method of paragraph A67, the method furthercomprising electrically stimulating the organoid using the electrode.

Paragraph A69. The method of any of paragraphs A57 to A68, wherein theinstrument includes a light guide optically coupled to a light sourceand having an aperture at the end introduced into the chamber.

Paragraph A70. The method of any of paragraphs A57 to A69, wherein theinstrument includes a magnet located at the end introduced into thechamber.

Paragraph A71. The method of any of paragraphs A57 to A70, wherein theinstrument includes an Attenuated Total Reflectance (ATR) probe.

Paragraph A72. The method of any of paragraphs A57 to A71, wherein theinstrument includes an endoscope.

Paragraph A73. The method of any of paragraphs A57 to A72, wherein theinstrument is coupled to a source of pneumatic or hydraulic pressure.

Paragraph A74. The method of any of paragraphs A1 to A73, furthercomprising introducing a test compound into the chamber.

Paragraph A75. The method of any of paragraphs A1 to A74, wherein themethod includes forming a plurality of organoids in a correspondingplurality of chambers.

Paragraph A76. The method of paragraph A75, further comprising applyinga different treatment to each of the organoids.

Paragraph A77. The method of paragraph A76, wherein applying a differenttreatment includes introducing a different test compound into eachchamber of the plurality of chambers.

Paragraph A78. The method of paragraph A77, wherein each different testcompound is a potential anti-cancer drug.

Paragraph A79. The method of any of paragraphs A76 to A78, furthercomprising introducing one or more cells, optionally cancer cells, intoeach of the chambers.

Paragraph A80. The method of paragraph A79, wherein the one or morecells are introduced into the organoid in the chamber, optionally via aneedle, optionally wherein the needle pierces a top wall of the chamber.

Paragraph A81. The method of any of paragraphs A76 to A80, furthercomprising collecting data for each organoid of the plurality oforganoids to test the different treatments for an effect on theorganoids.

Paragraph A82. The method of paragraph A81, wherein collecting dataincludes imaging each organoid in situ in its respective chamber.

Paragraph A83. The method of paragraph A81 or A82, wherein collectingdata includes assaying, for an analyte, a respective fluid associatedwith each organoid.

Paragraph A84. The method of any of paragraphs A81 to A83, whereincollecting data includes removing each organoid from its chamber,optionally after removing the sealing member.

Paragraph A85. The method of paragraph A84, wherein collecting dataincludes imaging each organoid after removal from its chamber.

Paragraph A86. The method of paragraph A85, wherein collecting dataincludes physically sectioning each organoid after removal from itschamber; and, optionally, imaging of a plurality of sections produced byphysically sectioning.

Paragraph B1. A method of organoid culture and/or analysis, the methodcomprising: (a) forming an organoid inside a chamber, wherein an accesstube overlies the chamber, and wherein a bottom end of the access tubeis closed by a top wall region of the chamber; (b) supplying nutrientsto the chamber to feed the organoid; (c) forming an opening through thetop wall region; and (d) inserting an end of an instrument into thechamber from the access tube.

Paragraph C1. A method of organoid culture and/or analysis, the methodcomprising: (a) sealing an open side of a receptacle to create achamber; (b) forming an organoid inside the chamber; (c) supplyingsubstances/fluid to the chamber; and (d) capturing an image of at leasta portion of the organoid while the organoid remains enclosed by wallsof the chamber.

Paragraph D1. A device for culture and/or analysis of an organizedmulti-cellular structure (e.g., an organoid), the device comprising: (a)a body defining a receptacle and at least two reservoirs, the at leasttwo reservoirs overlying the receptacle and being in separate fluidcommunication with the receptacle via respective channels; and (b) asealing member bonded or bondable to the body at an open side of thereceptacle, to create a chamber for an multi-cellular structure.Paragraph D2. The device of paragraph D1, further comprising a scaffoldfor biological cells attached to a wall of the receptacle.

Paragraph D3. The device of paragraph D2, wherein the scaffold isincluded in a hydrogel.

Paragraph D4. The device of paragraph D3, wherein the hydrogel containsbiological cells.

Paragraph D5. The device of paragraph D4, wherein the biological cellsinclude stem cells.

Paragraph D6. The device of any of paragraphs D3 to D5, wherein thehydrogel contains deposits of a drug. Paragraph D7. The device ofparagraph D6, wherein the deposits can be opened by light-induceduncaging to release the drug from the deposits.

Paragraph D8. The device of any of paragraphs D1 to D7, wherein thereceptacle and the at least two reservoirs are formed integrally withone another.

Paragraph D9. The device of any of paragraphs D1 to D8, wherein the bodyis injection-molded as a single piece.

Paragraph D10. The device of any of paragraphs D1 to D9, wherein eachreservoir of the at least two reservoirs shares a wall with thereceptacle.

Paragraph D11. The device of any of paragraphs D1 to D10, wherein eachchannel extends through a wall that is located intermediate, andoptionally shared between, the corresponding reservoir and thereceptacle.

Paragraph D12. The device of any of paragraphs D1 to D11, furthercomprising a removable lid configured to cover an open top of eachreservoir of the at least two reservoirs.

Paragraph D13. The device of any of paragraphs D1 to D12, wherein thedevice is in a sterilized condition.

Paragraph D14. The device of any of paragraphs D1 to D13, wherein thesealing member is bonded to the body. Paragraph D15. The device ofparagraph D14, wherein an organoid is contained in the chamber.

Paragraph D16. The device of any of paragraphs D1 to D15, wherein thebody defines at least four reservoirs each disposed in fluidcommunication with the receptacle.

Paragraph D17. The device of any of paragraphs D1 to D16, wherein thesealing member is configured to provide a bottom window for imaging atleast a portion of an organoid contained in the chamber.

Paragraph D18. The device of any of paragraphs D1 to D17, wherein thebody provides a pair of lateral windows to permit illumination of atleast a portion an organoid contained in the chamber via either of thelateral windows.

Paragraph D19. The device of any of paragraphs D1 to D18, wherein thebody defines an access tube having an open top end and closed bottomend, and wherein a shared wall separates the chamber from the bottom endof the tube.

Paragraph D20. The device of any of paragraphs D1 to D19, furthercomprising a linear array of substantially identical units connected toone another, wherein one of the units includes the receptacle and the atleast two reservoirs.

The term “exemplary” as used in the present disclosure, means“illustrative” or “serving as an example.” Similarly, the term“exemplify” means “illustrate by giving an example.” Neither termimplies desirability nor superiority.

The disclosure set forth above may encompass multiple distinctinventions with independent utility. Although each of these inventionshas been disclosed in its preferred form(s), the specific embodimentsthereof as disclosed and illustrated herein are not to be considered ina limiting sense, because numerous variations are possible. The subjectmatter of the inventions includes all novel and nonobvious combinationsand subcombinations of the various elements, features, functions, and/orproperties disclosed herein. The following claims particularly point outcertain combinations and subcombinations regarded as novel andnonobvious. Inventions embodied in other combinations andsubcombinations of features, functions, elements, and/or properties maybe claimed in applications claiming priority from this or a relatedapplication. Such claims, whether directed to a different invention orto the same invention, and whether broader, narrower, equal, ordifferent in scope to the original claims, also are regarded as includedwithin the subject matter of the inventions of the present disclosure.Further, ordinal indicators, such as first, second, or third, foridentified elements are used to distinguish between the elements, and donot indicate a particular position or order of such elements, unlessotherwise specifically stated.

1-29. (canceled)
 30. A device for culture of an organized multi-cellularstructure, the device comprising: (a) a body defining a receptacle andat least two reservoirs, the at least two reservoirs overlying thereceptacle and being in separate fluid communication with the receptaclevia respective channels; and (b) a sealing member bonded or bondable tothe body at an open side of the receptacle, to create a chamber for theorganized multi-cellular structure.
 31. The device of claim 30, furthercomprising a scaffold attached to a wall of the receptacle.
 32. Thedevice of claim 31, wherein the scaffold is included in a hydrogel. 33.The device of claim 30, wherein the receptacle and the at least tworeservoirs are formed integrally with one another.
 34. The device ofclaim 30, wherein the body is injection-molded as a single piece. 35.The device of claim 30, wherein each reservoir of the at least tworeservoirs shares a wall with the receptacle.
 36. The device of claim30, wherein each channel extends through a wall that is locatedintermediate the corresponding reservoir and the receptacle.
 37. Thedevice of claim 30, further comprising a removable lid configured tocover an open top of each reservoir of the at least two reservoirs. 38.The device of claim 37, wherein the lid comprises a flange configured torestrict lateral movement of the lid.
 39. The device of claim 37,wherein the lid comprises a fluid-tight seal with the at least tworeservoirs.
 40. The device of claim 30, wherein the device is in asterilized condition.
 41. The device of claim 30, wherein the sealingmember is bonded to the body.
 42. The device of claim 41, wherein thesealing member comprises at least one of a glass or a polymer.
 43. Thedevice of claim 30, wherein the body defines at least four reservoirseach disposed in fluid communication with the receptacle.
 44. The deviceof claim 30, wherein the sealing member comprises a bottom window forimaging a contents of an interior of the chamber.
 45. The device ofclaim 30, wherein the body comprises a pair of lateral windows to permitillumination of an interior of chamber via either of the lateralwindows.
 46. The device of claim 45, wherein the pair of lateral windowsare disposed opposite each other.
 47. The device of claim 30, whereinthe body defines an access tube having an open top end and closed bottomend, and wherein a shared wall separates the chamber from the bottom endof the tube.
 48. The device of claim 47, wherein the access tubecomprises a barrier at one end of the access tube.
 49. The device ofclaim 30, further comprising a linear array of substantially identicalunits connected to one another, wherein one of the units comprises thereceptacle and the at least two reservoirs.